Members of the genus Rickettsia cause a number of severe, vector-borne human diseases that include a variety of spotted fevers, as well as endemic, and epidemic typhus. These diseases pose a significant health threat worldwide. In addition, two species, R. rickettsii and R. prowazekii, the causative agents of Rocky Mountain spotted fever and epidemic typhus, respectively, are designated as re-emerging pathogens and Category B Select Agents due to their potential use as bioterrorist weapons. R. prowazekii is an obligate intracellular parasitic bacterium that invades eukaryotic cells and grows directly within the cytosol of the host cell, unbounded by a vacuolar membrane. The ability of R. prowazekii to exploit the intracytoplasmic environment and thereby cause serious human disease provides the momentum for our continuing studies. This proposal builds upon our studies that have established methods for the genetic manipulation of R. prowazekii and the isolation of mutants that allow for the testing of hypotheses regarding rickettsial virulence and intracellular growth. Specific targets of interest will include putative virulence-associated genes and secreted effectors involved in host-pathogen interactions. In Specific Aim 1 we will generate, isolate and characterize rickettsial mutants using both random transposon insertions and targeted mutagenesis techniques. To overcome a major barrier hindering rickettsial genetics, we will exploit our ability to generate fluorescent rickettsiae, and utilize our recent acquisition of a fluorescence activated cell sorter, to efficiently clone rickettsial mutants. Mutants will be generated in virulent strains so that virulence can be directly assessed. Additional targets will be selected based on their hypothesized importance to rickettsial intracellular survival. We will also explore methods for axenic growth of R. prowazekii. Mutant characterization will include growth and morphological assessments in selected cell lines and under a variety of culture conditions. Additionally, selected mutants will be assessed for virulence in the guinea pig model. In Specific Aim 2 we will examine the R. prowazekii secretome and its involvement in host-pathogen interactions. Putative effectors identified using functional screens for cell surface-exposed and secreted proteins will be added to a list of identified bioinformatic targets for validation of secretion. To demonstrate in vivo secretion of identified targets, tagged effectors will be expressed in R. prowazekii and localization confirmed. Secreted effectors will be expressed exogenously to evaluate interactions with host cell machinery. Genetic manipulation of R. prowazekii permits a gene-by-gene dissection of rickettsial functions and will have a significant impact on our understanding of R. prowazekii intracellular growth and pathogenicity.